Spectral analysis of the light proceeding from a specimen plays a very important role in many fields of specimen examination, in particular in fluorescence microscopy and in confocal scanning microscopy.
In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers, the track of the scanning light beam on or in the specimen ideally describing a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and stewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.). To permit acquisition of image data in layers, the specimen stage or the objective is displaced after a layer has been scanned, and the next layer to be scanned is thus brought into the focal plane of the objective.
German Unexamined Application DE 43 30 347 discloses an apparatus for the selection and detection of at least two spectral regions of a light beam, and the use of that apparatus in particular in a confocal scanning microscope. The apparatus has a selection device and a detection device, and for reliable simultaneous selection and detection of different spectral regions at high yield and with a simple design is configured in such a way that the selection device encompasses means for spectral dispersion of the light beam and means on the one hand for blocking out a first spectral region and on the other hand for reflecting at least one portion of the unblocked spectral region, and the detection device encompasses a first detector arranged in the beam path of the blocked-out first spectral region and a second detector arranged in the beam path of the reflected spectral region.
German Patent DE 195 10 102 C2 discloses an arrangement for confocal fluorescence microscopy in which an objective arrangement for acquiring an image of a specimen to be examined, at least one scanner mirror arranged after the objective arrangement, a tube lens, a confocal strip aperture, a first spectrometer arrangement, a wavelength selection aperture for selection of the emission wavelength, a second spectrometer arrangement identical to the first spectrometer arrangement, and a detector for acquiring the brightness distribution are arranged one behind another in an image beam path, an incoupling of excitation light being accomplished, backwards through the wavelength selection aperture, via an excitation beam path that leads from a source for monochromatic excitation light, through a strip aperture corresponding to the confocal strip aperture and a third spectrometer arrangement identical to the first spectrometer arrangement, to the wavelength selection aperture, the image beam path and the excitation beam path being coordinated with one another and constituted in such a way that light emitted from the specimen arrives via the wavelength selection aperture at the detector, but excitation light is prevented by the wavelength selection aperture from striking the detector but passes through the confocal strip aperture.
German Unexamined Application DE 198 42 288 A1 discloses an apparatus for adjustable detection of specimen light coming from an illuminated specimen, preferably in a microscope beam path, comprising at least one dispersive element for wavelength separation of the specimen light as well as means, arranged in the wavelength-separated portion of the specimen light, for adjustable blocking out of at least one wavelength region and deflection in the direction of at least one detector.
The known methods and apparatuses are of only limited suitability for spectral analysis of the light proceeding from a specimen and for the acquisition of entire spectra that are e.g. several tens or several hundreds of nanometers wide, and exhibit critical disadvantages for these applications. The apparatuses in which spectrometers, in particular grating spectrometers, are provided often have insufficient spectral resolution especially for fluorescence microscopy applications. This is usually attributable to unavoidable scattering of light at the grating structures. Apparatuses that contain prism spectrometers having downstream line detectors, for example photodiode arrays, exhibit insufficient dynamics in terms of detection sensitivity. The apparatuses that exhibit both good spectral resolution and good dynamics in terms of detection sensitivity, for example the apparatus known from the aforesaid German Patent DE 195 10 102 C2, are capable, however, of simultaneously detecting only a limited number of spectral regions to be defined by the user.